S-adenosyl-L-homocystein hydrolase promoter

ABSTRACT

A promoter derived from an SHH gene, especially the SHH gene of Arabidopsis thaliana which is capable of directing expression on a variety of operator genes in both monocotyledonous and dicotyledonous plants. The promoter of the invention may be used for directing expression of pathogen resistance genes to disease or wound sites.

This application is a 371 of PCT/GB/00882 filed Apr. 10, 1996.

FIELD OF THE INVENTION

The present invention relates to a promoter sequence capable of giving ahigh level of expression within plant cells. In particular, it relatesto a promoter derived from a gene encoding S-adenosyl-L-homocysteinehydrolase (SHH).

BACKGROUND OF THE INVENTION

Promoters control the spatial and temporal expression of genes bymodulating their level of transcription. Early approaches to geneticallyengineered crop plants utilised strong constitutive promoters to drivethe expression of foreign genes. As strategies in plant biotechnologyhave become more sophisticated, specific promoters have been used totarget transgene expression to a particular tissue or to a particulardevelopmental stage. The promoter of the present invention is especiallyversatile as it can be used either to give constitutive expression of agene or to target increased levels of gene expression at sites ofwounding or pathogen invasion.

SHH was first described, in rat liver extracts, as the activityresponsible for the reversible hydrolysis of S-adenosyl-L-homocysteine(SAH) to adenosine and homocysteine by the cleavage of a thioether bondin SAH [de la Haba, G. and Cantoni, G. L. (1959). J. Biol. Chem. 234,603-608].

SAH is formed as a direct product of transmethylation reactionsinvolving S-adenosyl-L-methionine (SAM) [Cantoni, G. L. and Scarano, E.(1954). J. Am. Chem. Soc. 76, 4744] and is known to be a potentinhibitor of most SAM mediated methyltransfer reactions. Therefore SAHis converted to homocysteine and adenosine by SHH as shown schematicallybelow:

    ______________________________________                                        S-adenosyl-L-methionine (SAM)                                                             ↑↓ Methyltransferase                                 Methylated Product + S-adenosyl-L-homocysteine (SAH)                                           ↑↓ SHH                                                    Adenosine + L-homocysteine                                                             ↑↓ N5-methyltetrahydrofolate                                        Methionine                                              ______________________________________                                    

This pathway for the metabolism of SAH is the only pathway in mostspecies. SHH has been found in all cells tested with the exception ofEscherichia coli and other related bacteria [Shimzu, S. et al. (1984).Eur. J. Biochem. 141, 385-392].

The unique metabolic role of SHH in the removal of SAH and thestructural complexity of the enzyme suggest that SHH may have a role inthe regulation of the biological utilisation of SAM. SAM serves as amajor methyl group donor for numerous highly specific methyltransferasereactions with a large variety of acceptor molecules; for examplephenylpropanoid derivatives, cyclic fatty acids, proteins,polysaccharides and nucleic acids [Tabor, C. W. and Tabor, H. (1984).Adv. Enzymol. 56, 251-282]. It should be noted that SAM also hasregulatory functions, namely the allosteric stimulation of threoninesynthase. In plants, SHH has been studied primarily in relation to thebiosynthesis of various phenylpropanoid derivatives.

Enzymes affecting the intracellular levels of SAH are important in thestudy of plant methylation reactions because it has been demonstratedthat many methyltransferases are inhibited by SAH [Deguchi, T. andBarchos, J. (1971). J. Biol. Chem. 246, 3175-3181]. For example, anenzyme catalysing the methylation of caffeic acid was purified fromspinach-beet leaves and found to be potently inhibited by SAH [Poulton,J. E. and Butt, V. S. (1976). Arch. of Biochem. Biophys. 172, 135-142].Other metabolic pathways of the plant which involve transmethylation arethe production of lignin and suberin, which are both derived fromphenylalanine, through a series of reactions. These reactions includethe methylation of caffeic acid into ferulic acid and also themethylation of s-hydroxyferulic acid into sinapic acid. Both thesemethylation reactions require SAM and hence produce SAH as a byproductwhich needs to be removed by SHH to allow further transmethylation.

Once SHH had been isolated, many factors were calculated, such as theenzyme's pH optimum of 8.5, with a 50% activity between pH 6.5-10. Dueto the Km value found for the substrate, L-homocysteine, the synthesisof SAH proceeds in vivo at a significant rate only when L-homocysteineis accumulated [Poulton, J. E. and Butt, V. S.(1976). Arch. of Biochem.Biophys. 172, 135-142].

In vivo, the adenosine produced by the hydrolysis of SAH is notdeaminated but is converted to ADP by the successive action of adenosinekinase and adenylate kinase, both of which enzymes have beendemonstrated in spinach-beet leaves. If L-homocysteine accumulates, itcauses inhibition of SHH activity and therefore in vivo, L-homocysteineappears to be methylated by N5-methyltetrahydrofolate to methionine.Indeed, this reaction has been demonstrated in pea seedling extracts andspinach and barley leaves. Unlike all animal SHH enzymes, plant SHH isnot inhibited by adenosine but is instead stabilised by lowconcentrations [Jakubowski, H. and Guranowski, A. (1981). Biochem. 20,6877-6881].

The kinetic evidence shows that SHH is a sensitive regulator of SAHutilisation, its activity depending not only upon favourableconcentrations of metabolites in relation to equilibrium conditions butalso upon the levels of SAM, adenosine and L-homocysteine maintainedwithin the system. These in turn will act as feed back inhibitors oractivators to determine the rate of methylation reactions which aresensitive to the levels of SAH [Poulton, J. E. and Butt, V. S. (1976).Arch. of Biochem. Biophys. 172, 135-142].

As previously mentioned SHH has been found in all organisms testedexcept E. coli and some related species where a two step enzymaticprocess hydrolyses SAH into adenosine and L-homocysteine. So far thefollowing SHH cDNAs have been isolated and published:

Rat [Ogawa, H. et al. (1987). Proc. Natl. Acad. Sci. USA. 84, 719-723],

Dictostelium discoideum [Kasir, J. et al. (1988). Biochem. Biophys. Res.Commun. 153, 359-364]

Human [Coulter-Karis, D. E. and Hershfield, M. S. (1989). Ann. Hum.Genet. 53, 169-175]

Caenorhabditis elegans [Prasad. S. S. et al. (1991). EMBL databaseAccession No. M64306]

Leishmania donovani [Henderson, D. M. and Ullman, B. (1992). EMBLdatabase Accession No. M76556]

Petroselinum crispum [Kawalleck, P. et al. (1992). Proc. Natl. Acad.Sci. USA. 89, 4713-4717]

Rhodobacter capsulatus [Sganga, M. W. et al. (1992). Proc. Natl. Acad.Sci. USA. 89, 6328-6332]

The high level of homology between SHHs of evolutionary divergentspecies was highlighted further following isolation of SHH from the rat,from Dictostelium discoideum, from the purple non-sulphur photosyntheticbacterium Rhodobacter capsulatus and then from parsley (Petroselinumcrispum). The bacterial SHH shows a remarkable degree of amino acidsequence homology, approximately 65% identity and 77% similarity to thepreviously isolated SHHs from rat, D. discoideum, human and C. elegans.This is one of the highest levels of sequence conservation ever reportedbetween proteins having a similar function in prokaryotes and humans.Similarly, SHH cDNA from parsley is 64% identical to rat cDNA and thereis 79% similarity at the amino acid level. The lack of sequencedivergence between species may suggest a stringent requirement for SHHto retain its primary structure for function.

Both the R. capsulatus and the parsley amino acid sequences have anadditional amino acid motif in comparison to the rat, D. discoideum,human, C. elegans and L. donovani sequences. R. capsulatus has anadditional 36 amino acid region whereas parsley has an additional 41amino acids. These two additional stretches are found in the sameposition in the predicted protein sequence, approximately one-third ofthe distance from the amino terminus. (see FIGS. 3A-3C) although they donot show significant homology.

SUMMARY OF THE INVENTION

The present inventors have now isolated SHH from various other plantsources. The first of these was Asparagus officinalis and the nucleotidesequence and deduced amino acid sequences for this (SEQ ID NO 1 and SEQID NO 2) together with the positions of the restriction sites are shownin FIGS. 1A-1F.

Asparagus SHH also contains the extra stretch of residues earlier foundin the other photosynthetic species, parsley and R. capsulatus and notin SHH cDNAs from non-photosynthetic species. This 41 amino acidstretch, from amino acids 150 to 190 is as well conserved between thedicotyledon species parsley and the monocotyledon species asparagus asis the rest of the sequence although it is not similar to the 36 aminoacid stretch from R. capsulatus. This is illustrated in FIGS. 2A, 2B,3A-3C.

Following this, SHH cDNAs were also isolated from other species and oneof the species selected was Arabidopsis thaliana. The promoter derivedfrom the SHH gene from A. thaliana has proved to be particularly usefulas it directs a high level of expression of a variety of genes,exemplifed by the reporter genes glucuronidase (GUS) and luciferase(LUC). Promoters from the SHH genes of other species may also beisolated using the same techniques and may also be expected to haveuseful and advantageous effects.

Therefore, in a first aspect of the present invention, there is provideda promoter derived from an SHH gene.

It is preferred that the SHH gene is that derived from A. thaliana.

The promoter has several useful properties and, in particular, becauseof the uniformity of the SHH gene over different species, it is capableof directing the expression of a wide variety of effect genes in plants,particularly crop plants such as Arabidopsis, tobacco, oil seed rape,potato, tomato, banana, wheat and maize.

The sequence of the Arabidopsis promoter (SEQ ID NO 3) is shown in FIGS.5A-5C and thus in a second aspect of the invention, there is provided apromoter having the sequence of SEQ ID NO 3 or a sequence of at least70% homology thereto.

It is preferred that the sequence of the promoter has not less than 80%homology, and, more preferably 90% homology to SEQ ID NO 3.

Since transmethylation reactions are important components of thebiosynthetic machinery in most plant cells, the SHH will be expressed incells throughout the plant. The promoter derived from the SHH willtherefore provide a useful control mechanism for expression of anyeffect gene in a constitutive manner. The effect gene may be an SHH genebut will more usually be an introduced gene. Examples of introducedeffect genes which may be linked to the promoter of the presentinvention include selectable markers such as NptII, the kanamycinresistance gene, the phosphinothricin resistance gene or thephosphinothricin acetyl transferase (PAT) gene and others such as theglucuronidase (GUS) and luciferase (LUC) reporter genes.

The predicted increase in transmethylation and concomitant increase inSHH activity following wounding or pathogen invasion means that the SHHgene will also be useful in providing increased levels of expression ofintroduced genes at sites of wounding and pathogen invasion. In thisrespect, the SHH promoter will be particularly useful for targetingexpression of disease resistance genes, for example genes encodingantifungal proteins such as those described in our earlier patentapplications published as WO92/15691, WO92/21699 and WO93/05 153. Usingthe SHH promoter, these antifungal proteins can be targeted to woundsites to prevent fungal invasion or to sites of infection to preventfurther spread of the pathogen. The combined constitutive andwound/pathogen induced expression will thus provide a powerful mechanismfor the prevention of disease using introduced genes.

In order to direct expression, the promoter and its associated effectgene must, of course be incorporated into a vector and therefore, in afurther aspect of the invention there is provided a vector comprisingthe promoter of the present invention linked to an effect gene.

For expression in dicotyledonous plants binary agrobacterium vectors areparticularly suitable whereas for monocotyledonous plants direct DNAdelivery vectors are preferred.

As already mentioned above, the sequence of the SHH gene is conserved toa remarkable extent between species. The promoter of the presentinvention can therefore be used to direct expression in almost any plantspecies, whether monocotyledonous or dicotyledonous. It is of particularuse in crop species such as wheat, maize, oil seed rape, potato, tomato,banana and tobacco.

Thus in a further aspect of the invention there is provided a plant celltransformed with a vector as described above. Transformation may beacheived by standard techniques.

The invention also provides a genetically transformed plant and partsthereof, such as cells protoplasts and seeds, having stably incorporatedinto the genome the construct of the present invention. Any plant may bechosen but the crop species listed above are particularly preferred.

As already mentioned, the expression of SHH at disease or wound sitesmeans that the promoter will be of particular use in combating diseasewhen linked to an appropriate effect gene.

Therefore, in a further aspect, the invention provides a method ofincreasing the resistance of a plant to infection by a pathogenicorganism, the method comprising transforming the plant with a vectorcomprising a promoter according to the first aspect of the inventionoperably linked to a gene conferring resistance to the pathogenicorganism.

Examples of genes conferring resistance to pathogenic organisms includethe genes encoding antifungal proteins described in WO92/15691,WO92/21699 and WO93/05153.

The isolation of the promoter of the present invention was achieved as aresult of the study of SHH in various plant species. The strategyemployed was firstly to isolate the gene encoding asparagus SHH. Thisconfirmed the remarkable degree of sequence identity in the SHH genebetween plant species and was used as a basis for the design ofpolymerase chain reaction (PCR) primers which were used to isolate SHHgenes from various other plant species including Arabidopsis thaliana.Analysis of the A. thaliana SHH gene revealed a 1849 base promoter, thepromoter of the present invention, which, further experimentationdemonstrated to be a highly versatile promoter capable of directingexpression of different genes in a variety of plant species.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described for the purposes ofillustration only with reference to the following examples and to thefigures in which:

FIGS. 1A-F shows the nucleotide and deduced amino acid sequence ofasparagus SHH (SEQ ID NOS 1 and 2). In FIGS. 1A-1F, the @ symbols definethe positions of the start and finish of the original DB6 clone; thesites indicated were used for the sub-cloning of DB6 and the primersused in PCR experiments are underlined.

FIGS. 2A-2B comprise a comparison of full length predicted SHH proteinfrom asparagus (Dbf) (SEQ ID NO 2) with SHH protein from parsley(Pcshh), the NAD⁻ binding site has been underlined in all species.

FIGS. 3A-3C comprise a comparison of SHH predicted amino acid sequencefrom asparagus (Dbf) with SHH proteins from rat, parsley (Pcshh), R.capsulatus (Rcahcy) and C. elegans (Cehcg); the NAD⁻ binding site hasbeen underlined in all species and * denotes amino acids conserved inall species.

FIG. 4A shows the amino acid sequence alignment of cloned PCR products(without the primers) from asparagus (ASP, SEQ ID NO 2), Arabidopsis(ARA, SEQ ID NO 4), tobacco (TOB, SEQ ID NO 5), Brachypodium (ERA, SEQID NO 6) and wheat (WH and WHU, SEQ ID NOS 7 and 8). The * denote aminoacids conerved in every species and denotes conservative amino acidchanges.

FIG. 4B is the same as FIG. 4A but with the smaller wheat productremoved to highlight sequence conservation between the other five PCRspecies.

FIGS. 5A-5C shows the SHH promoter sequence from Arabidopsis thaliana(SEQ ID NO 3) including the first 30 amino acids used in translationaltransgene fusions.

FIGS. 6A-6K comprise a map of the A. thaliana gene showing codingsequence, intron and 3' untranslated region. Important restriction sitesare also shown.

FIGS. 7A-7D show the coversion of plasmid pSK AoPR1 FULL LUC via pSKAtSHH LUC to pBI101 At SHH Luc and pSK AtSHH-GUS to pBIN 19 AtSHH GUSand pBI101 At SHH Correct to pBI101 At SHH Wrong.

FIG. 8 shows a comparison of SHH driven LUC activity in stem sectionsand wounded leaf in tobacco.

FIG. 9 shows SHH driven LUC expression in various tissues in tobacco.

FIG. 10 shows LUC line 11 wounding time course in tobacco.

FIG. 11 shows LUC line 11; open flower non dehisced in tobacco.

FIG. 12 shows SHH driven GUS expression during A. thaliana seedlingdevelopment.

FIG. 13 shows SHH driven GUS expression in various A. thaliana tissues.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 Elucidation of cDNASequence of Asparagus S-Adenosyl-L-Homocysteine Hydrolase

This research utilised previously constructed cDNA libraries derivedfrom an mRNA population purified from mechanically separated Asparagusofficinalis cells that had been prepared from asparagus seedlings bygrinding in a mortar and pestle [Paul, E. et al. (1989), Plant Science,65, 111-117 and Harikrishna, K., et.al. (1991), Journal of ExperimentalBotany, 42, 791-797].

Clones were randomly picked from the existing cDNA library made usingmRNA extracted from model system cells 1-3 days after mechanicalisolation and short stretches of derived sequence data. This data wasanalysed using Pearson and Lipman searches for homologous knownsequences within the EMBL database. A putative asparagus SHH cDNA wasidentified in this manner and called DB6. This clone was subcloned andthe full sequence was derived. The positions of the restriction sitesused for this purpose are shown in FIGS. 1A-1F.

The nucleotide sequence itself and the translation of this deduced 1633bp sequence were compared to the published SHH clones (particularly theparsley SHH), which demonstrated that DB6 was not full length, with 11amino acids being absent from the amino terminus. Therefore existinglibraries were rescreened using the DB6 insert as a probe and a fulllength version isolated (SEQ ID NO 1). Interestingly this version, namedDBF, was isolated from a different library from the original clone. DB6was picked from a day 1-3 library whereas DBF was isolated from a day 1library. Sequence data revealed DBF to encode the full SHH amino acidsequence of 485 residues (SEQ ID NO 2), with 25 bp of 5' untranslatednucleotides, 284 bp of 3'-untranslated nucleotides and a polyA+ tail.

Genomic Southern data has shown that the asparagus SHH is probably amember of a small gene family, as was found with the parsley homolog. Aswith the parsley SHH, the asparagus SHH has been isolated from a modelsystem. However, whereas a fungal elicitor was added to the culturedparsley cells, the asparagus system does not use elicitor treatment andrelies on gene induction due to the mechanical isolation of the cells,and therefore it aims to isolate wound induced genes.

FIGS. 2A, 2B and 3A-3C show the asparagus SHH also contains the extrastretch of residues found in the photosynthetic species parsley and R.capsulatus and not in the other cloned SHH cDNAs from non-photosyntheticspecies. This 41 amino acid stretch, from 150-190 amino acids is as wellconserved between the dicotyledon parsley and the monocotyledonasparagus as is the rest of the amino acid sequence, unlike the 36residue stretch of R. capsulatus.

EXAMPLE 2 Isolation of SHH Genes from Other Plant Species andDemonstration of Sequence Conservation

To enable further studies as to the significance of this `extra` regionin photosynthetic organisms amino acid sequence of SHH, PCR (PolymeraseChain Reaction) primers were designed to either side of the 41 aminoacid stretch common to parsley and asparagus SHH. The primers designedwere the following and are shown in context of the SHH cDNA in FIGS.1A-1F:

PCR-1 (SEQ ID NO 10)

5' GCGTCTAGATGCAACATACTTCTCCAACCTAGGA 3'

PCR-2 (SEQ ID NO 11)

5' GCGTCTAGATTAGTCAAACTTGCTCTTGGTAGAC 3'

It was expected that a PCR product of 482 bp would be produced incontrol experiments with asparagus genomic DNA as the template, unlessan intron existed between the primer annealing sites in the genomicgene. The possibility of introns between the primer binding sites wasruled out following a PCR experiment showing that the expected 482 bpproduct was obtained. Of this 482 bp product, 63 bp consist of primersequence (31 bp+32 bp). The first 9 bp of each primer, at the 5' end,were designed with an XbaI site to facilitate cloning of PCR products.

These PCR primers were used to try and amplify a segment of the SHH genefrom several plant species whose DNA was available within thelaboratory. For all species tested, similar sized products wereobtained. When these products were hybridised to the asparagus SHH cDNAprobe good hybridisation was observed. SHH PCR products were amplifiedfrom Arabidopsis, Asparagus (as a control), Tobacco, Brachypodium andWheat. A single 480 bp PCR product was produced from the Arabidopsis,Asparagus and Brachypodium experiments; whereas wheat and tobacco bothproduced further products of 350 bp and 700 bp respectively, in additionto the predicted size product. In all cases a product of the predictedsize was found. The second tobacco product of 700 bp was later proved tobe this size due to multimers of PCR-2 primer sequence on one end, as aresult of ligation or PCR error.

The other wheat product was smaller than predicted (350 bp) and when itwas cloned and sequenced it was revealed why this was the case. Initialattempts to clone the PCR products into pBluescript (Trade Mark) usingthe XbaI site within the primers failed except for the control productfrom asparagus. Therefore a commercial vector available specifically forthe cloning of PCR products was used, this vector is called PCRII. Thevector utilises the fact that Taq polymerase used in PCR will add singledeoxyadenosines to the 3'-end of all duplex molecules, thereforeeliminating the need for restriction sites within the primers. All thePCR products from each species mentioned were cloned in this manner andthen sequenced. This sequence data revealed why the initial attempts atcloning into pBluescript had failed. During the PCR reaction, for anunknown reason, the whole primer had not always been replicated at its5'-end, causing the recognition site of XbaI not to be present in thefinal product. In most cases one primer had the site while the other didnot.

All the clones were sequenced and multiple line-ups performed as can beseen in FIG. 4 which compares the deduced amino acid sequences forasparagus (SEQ ID NO 1), A. thaliana (SEQ ID NO 4), tobacco (SEQ ID NO5), Brachypodium (SEQ ID NO 6) and the two wheat products (SEQ ID NOS 7and 8). The smaller of the two wheat products proved to be more closelyrelated to the nonphotosynthetic cDNAs isolated, in that it did notcontain the extra stretch of 41 amino acids found in parsley andasparagus. The validity of this product needs to be checked as it mayhave arisen through contamination. Computer analysis has already proventhis not to be the same as the Human SHH, previously cloned. However asa 480 bp wheat product was also cloned this could enhance the argumentthat SHH genes exist as small gene families encoding enzymes withdiffering biological/physiological roles.

In summary these data shows the SHH gene sequences to be highlyconserved across the plant kingdom for the following reasons; firstly,the PCR primers facilitated the successful amplification of the SHHsequence from every tested plant species and secondly, the actualnucleotide and predicted amino acid sequence of this region shows howconserved the SHH gene is between plant species spanning themonocotyledon/dicotyledon classification. (See FIG. 4).

Thus it has been shown that the SHH amino acid sequence is highlyconserved between a diverse range of plant species.

EXAMPLE3 Demonstration of the Role of SHH in Transmethylation Reactions

Molecular and Biochemical Characterisation

It was predicted that an accumulation of SAH would inhibit the SAMmediated caffeic acid-O-methyltransferase reaction.

If, as suggested, SHH has a central role in allowing thetransmethylation reactions of several metabolic pathways to occurunhindered it must be present and active in specific regions of theplant at specific developmental periods. Therefore the well studiedlignification process occurring in the stems of maturing tobacco wheretwo well characterised transmethylation reactions occur in thebiosynthesis of lignin precursors would confirm the point. Although SHHtranscript levels may vary between organs, for example lignifying stems,leaves, roots, pollen etc., it does not necessarily mean that theactivity of the enzyme will be altered.

To examine the expression of the SHH gene in a range of tobacco organs,steady state mRNA levels were determined using northern analysis andenzyme assays were used to determine the level of enzyme activity.

Northerns were performed using standard techniques with the tobacco PCRproduct (FIG. 4) or cDNA as a hybridisation probe. Extraction of SHHenzyme and assay of activity were performed as follows:

All extraction steps were performed at 4° C.

1. Homogenise plant tissue (˜1 g) by grinding in a pestle and mortarwith 2 v/w extraction buffer [100 mM Tris pH8, 10 mM SodiumMetabisulphite, 10 mM Ascorbic Acid and 5 mM DTT added on day of use],acid washed sand and 0.1 g of insoluble PVP.

2. Decant the supernatant and centrifuge at 17000 g for 15 min. Removethe supernatant, noting the volume and add 0.56 g of solid ammoniumsulphate per ml. Stir for 30 min.

3. Centrifuge at 17000 g for 15 min, resuspend the pellet in 2.5 ml ofresuspension buffer [100 mM Tris pH8 and 5 mM DTT added on day of use]and clarify the solution by pulse centrifugation.

4. The extract is then desalted on a Pharmacia PD-10 G-25 column whichhas been pre-equilibrated with resuspension buffer according to themanufacturer's protocol. The resultant eluate is used in the followingassay procedure.

5. Sequentially add the following to a microcentrifuge tube

a) 10 μl of 100 mM DL-Homocysteine

b) 80 μl of enzyme extract

c) 10 μl of Adenosine (100 μl of 53 mCi/mmol ¹⁴ C-adenosine and 100 μlof 20 mM adenosine)

6. Mix and incubate at 300° C. for 30 min.

7. Stop the reaction by adding 10 μl of 50% TCA and stand on ice for 10min.

8. Centrifuge and apply 20 μl to a 1.5 cm wide strip on a silica TLCplate containing fluorescent indicator (F254, Merck). Develop the platefor a distance of 10 cm in butan-1-ol+acetic acid+water (12:3:5).

9. After allowing the plate to dry, visualise the SAH product with a UVlamp at 254 nm. Cut out these areas and elute the silica from the platewith 0.5 ml methanol before scintillation counting.

Northern analysis showed the SHH transcript to be detected at very lowlevels in most tissues tested. SHH enzyme assays demonstrated thattranscript levels and enzyme activity levels do correlate strongly.Inducible SHH enzyme activity was found in wounded tissue fromasparagus, tobacco and Arabidopsis when compared with SHH enzymeactivity in unwounded tissue. The products of the enzyme assay wereseparated on a TLC plate according to Poulton and Butt (Archives ofBiochemistry and Biophysics 172, 135-142, 1976) and both ¹⁴ C labelledadenosine and S-adenosyl homocysteine were detected. The rf values ofboth ¹⁴ C labelled compounds compared favourably with those obtained forunlabelled sources that were run on the plates simultaneously anddetected by UV fluorescence. In the absence of homocysteine or enzymepreparation, no fluorescent products were observed with the same rfvalues as unlabelled SAH. These data demonstrate that ¹⁴ C labelled SAHwas derived from the catalytic conversion of ¹⁴ C labelled adenosine andhomocysteine by the SAH enzyme present in the plant preparations.

EXAMPLE 4 Isolation of a SHH Gene from Arabidopsis thaliana

The PCR fragment of the SHH gene from Arabidopsis was used to screen anArabidopsis genomic library for the corresponding gene using standardtechniques. Positive clones arising from the screen were analysed andthe SHH gene sequenced from a candidate clone containing the gene andits promoter control regions. The DNA sequence of the promoter is shownin FIGS. 5A-5C and the DNA and deduced amino acid sequence of the codingregion in FIGS. 6A-6K.

EXAMPLE 5 SHH Gene Down-Regulation and Over-Expression Studies

The Arabidopsis gene sequence described above was used in a series ofexperiments to modulate SHH gene activity either by down-regulationusing antisense or partial sense constructs or by over-expression usingthe full coding sequence thus reducing the increasing SHH enzymeactivity respectively. Effects in specific plant organs or at particularsites of metabolism may be achieved through use of appropriate genepromoters; for example, the lignification process may be modified byusing a gene promoter isolated from a gene specific to lignifyingtissues such as cinnamoyl:CoA reductase or cinnamyl alcoholdehydrogenase. Alternatively, specific organs may be targeted such asthe anthers using the Arabidopsis A9 or APG promoters or pollen usingthe maize ZM13 promoter. Furthermore gene activity could be modified atsites of pathogen attack or wounding through use of wound promoter e.g.AoPRI from asparagus. Finally, SHH enzyme activities may be modifiedthroughout the plant by using a promoter expressed in most plant tissuese.g. CaMV 35S.

EXAMPLE 6 Analysis of Arabidopsis SHH Promoter Activity

The promoter isolated from the Arabidopsis SHH gene has been tested intransgenic tobacco plants and in A. thaliana to establish its pattern ofexpression. As shown below this promoter has high level expression inall organs analysed and an additional activity which is inducedfollowing wounding. It therefore has utility as a constitutive promoterfor expression of selectable markers for in vitro selection oftransformants or for high level expression in mature plants.Furthermore, the wound induced activity may be used for directing geneproducts (e.g. antifungal proteins) to sites of wounding or pathogeninvasion. Construction of the SHH promoter--reporter gene wereundertaken as follows:

1. Transcriptional fusions between the SHH promoter and the luciferase(LUC) reporter gene.

The following construct is based on pSK AoPR1-LUC as describedpreviously (Warner et al. The Plant Journal 6:31-43,1994). Thisconstruct (FIG. 7) was digested with NcoI and XhoI to remove the AoPRIpromoter. Using these sites the Arabidopsis SHH promoter was ligatedinto the plasmid in front of Luc via an NcoI site to create pSKAtSHH-LUC (FIGS. 7A-7D), a cloning intermediate.

The binary vector pBI01 AoPR1-LUC (Warner et al., 1994) was disgestedwith BamHI and SalI to remove the AoPRI-LUC cassette and theXhoI/BamHI-digested SHH promoter-LUC reporter cassette from pSKAtSHH-LUC (FIGS. 7A-7D) was ligated into the plasmid to create pBI01AtSHH-LUC (FIG. 7B).

2. Transcriptional fusions between the SHH promoter and theglucuronidase (GUS) reporter gene.

Similar SHH promoter-reporter cassettes were constructed utilising theGUS reporter in place of the LUC reporter. This facilitated directcomparisons between the two reporters under the control of the sameArabidopsis SHH promoter.

Initially a pSK-derived plasmid containing a NOS terminator behind theGUS gene containing an NcoI site at the initiating methionine codon wasdigested with NcoI/XhoI. The Arabidopsis SHH promoter was similarlydigested and ligated into the vector to create pSK AtSHH-GUS (FIG. 7C).The XhoI/BamHI fragment of this plasmid was then cloned into theBamHI/SalI sites of BIN19 (Bevan, M. (1984), Nucleic Acids Research, 12,8711-8721) to create a binary plasmid pBIN19 AtSHH-GUS (FIG. 7C).

3. Translational fusions between the SHH promoter and the glucuronidase(GUS) reporter gene.

A simple one step cloning process allowed a further GUS fusion to bemade using pBI01. From sequence data it was predicted that a fusion tobe made using pBI01 would generate an active transitional fusion betweenthe Arabidopsis SHH promoter and GUS with the first 30 amino acids ofthe GUS fusion encoded by the SHH gene. This construct was made byligating the 1949 bp XhoI fragment of the SHH promoter into the SalIsite of pBI01. The resultant clone was named pBI01.1 in the oppositeorientation (i.e. in the anti-sense orientation) creating pBI01 AtSHHWrong (FIG. 7D). This construct (FIG. 7D) was used as a negative controlin expression studies.

REPORTER GENE ASSAYS

GUS activity was determined using standard techniques (Jefferson). LUCassays were performed essentially as in Ow et al. Science, 234, 856-859,1986 with modifications described by Warner et al., 1994.

FIGS. 8-11 show luciferase activity data expressed as light units/μgtotal protein for one representative transgenic tobacco line. Identicalreporter expression patterns were observed in several other SHHpromoter-LUC and SHH promoter-GUS tobacco transgenic lines.

Similar patterns of reporter gene expression were also observed withintransgenic A. thaliana, as demonstrated in FIGS. 12 and 13. These A.thaliana transgenics represent T3 homozygous lines containing a singlecopy T-DNA. Fluorometric assays of GUS activity within leaves of severalfo these lines prove that the exprssion due to the SHH promoter occursat levels similar to or greater than CaMV35s-driven GUS levels insimilar transformants. Of seventy-one individual transformed linesharbouring the pBI121 [Jefferson et al (1987) EMBO J., 6, 3901-3907],the highest activity within leaves was found to be 12040 pmol MU/min/mg,with an average between 2000 and 3000 pmol MU/min/mg [Clarke et al,(1992) Plant Mol. Biol. Reporter, 10, 178-189]. Of the five chosenSHH-GUS A. thaliana homozygous T3 lines, the expression within leavesvaries from 20984 pmol MU/min/mg to 4420 pmol MU/min/mg with an averageof 13725 pmol MU/min/mg, a greater value than the highest expressingline using pBI121.

Histochemically stained transgenic tobacco tissues supported theexpression data for GUS activity in all tissues tested.

These results show that the AtSHH promoter drives reporter geneexpression in all tissues tested. The point of interest lies in therespective levels of the expression. AtSHH promoter reporter geneexpression levels in transgenic plants were far higher than would bepredicted from the levels of endogenous SHH transcript. The results intobacco may be explainable in terms of aberrant expression driven by theArabidopsis promoter in the tobacco host plant due to incorrecttranscription factors recognising the introduced promoter but theincreased levels of expression in A. thaliana suggest that this is notthe case. Alternatively, the high levels of reporter gene activity couldbe a result of stabilisation or high levels of translation of thereporter gene transcript affected by the Arabidopsis SHH 5' leadersequence present in all constructs made.

The AtSHH promoter, has been demonstrated to cause increased reportergene expression in tobacco and in A. thaliana, and this demonstrates itsutility as a high level constitutive promoter.

Furthermore, superimposed on the constitutive expression pattern of theAtSHH promoter is a 2.5-fold increase in expression at wound sites whichcan be clearly seen in FIG. 10.

EXAMPLE 7

To establish utility of the Arabidopsis SHH promoter in directingexpression of an ATP gene and providing resistance to a fungal pathogen,the 1760 bp promoter fragment from pSKAt SHH-GUS was amplified by PCRusing the primers to change the 5' XhoI site to HindIII and the NcoIsite at the ATG start codon to XhoI. The resulting fragment was cloneddirectly into a pMJB1 vector as a partial HindIII-XhoI fragment suchthat the promoter is placed upstream of the ATP gene. An omegatranslational enhancer from tobacco mosaic virus, located between theSHH promoter and the ATP gene is included to increase the level of geneexpression. In this example, the ATP gene is Dm-AMP1 obtained from seedsof Dahlia merckii. The resulting construct was introduced into oilseedrape using standard Agrobacterium-mediated transformation techniques.Transformed plants wre screened for expression of the Dm-AMP1 gene usingwestern blotting techniques and expressing lines advanced into detachedleaf disease assays with the oil seed rape pathogen Phoma lingam(Gretenkort and Ingram (1993), J. Phytopathology, 137, 89-104).Introduction of the Dm-AMP1 gene and expression by the SHH promoterresulted in increased resistance to infection by Phoma lingam. Theseobservations parallel those obtained when expression of the Dm-AMP1 geneis controlled by a well-known constitutive promoter, 35S, fromcauliflower mosaic virus, exemplifying the utility of the SHH promoterin this application.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1767 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: SHH GENE - #FROM ASPARAGUS                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION:26..1483                                                         (D) OTHER INFORMATION:/cod - #on.sub.-- start= 26                                  /product=- # "Asparagus SHH"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - CTCGTTTCAG ATCCGATCTG AAGAA ATG GCT CTC CTC GTT - #GAG AAG ACT ACC           52                                                                                         - #          Met Ala Leu Leu Val - # Glu Lys Thr Thr                          - #            1      - #         5                          - - TCT GGC CGC GAG TAC AAG GTC AAG GAC ATG TC - #T CAG GCC GAC TTC GGC          100                                                                       Ser Gly Arg Glu Tyr Lys Val Lys Asp Met Se - #r Gln Ala Asp Phe Gly            10                 - # 15                 - # 20                 - # 25       - - CGC CTC GAG ATC GAG CTC GCT GAG GTC GAG AT - #G CCA GGG CTC ATG GCC          148                                                                       Arg Leu Glu Ile Glu Leu Ala Glu Val Glu Me - #t Pro Gly Leu Met Ala                            30 - #                 35 - #                 40              - - TGC CGT GCC GAG TTC GGC CCC GCC CAG CCA TT - #C AAG GGC GCA AAA ATC          196                                                                       Cys Arg Ala Glu Phe Gly Pro Ala Gln Pro Ph - #e Lys Gly Ala Lys Ile                        45     - #             50     - #             55                  - - ACT GGA TCC CTC CAC ATG ACG ATC CAA ACT GC - #C GTC CTC ATC GAA ACC          244                                                                       Thr Gly Ser Leu His Met Thr Ile Gln Thr Al - #a Val Leu Ile Glu Thr                    60         - #         65         - #         70                      - - CTA ACC GCC CTC GGG CCC GAG GTT CGC TGG TG - #C TCC TGC AAC ATA TTC          292                                                                       Leu Thr Ala Leu Gly Pro Glu Val Arg Trp Cy - #s Ser Cys Asn Ile Phe                75             - #     80             - #     85                          - - TCC ACC CAG GAC CAT GCC GCC GCT GCC ATT GC - #C CGT GAC TCC GCC TCC          340                                                                       Ser Thr Gln Asp His Ala Ala Ala Ala Ile Al - #a Arg Asp Ser Ala Ser            90                 - # 95                 - #100                 - #105       - - GTC TTC GCC TGG AAG GGT GAG ACC CTC CAG GA - #G TAC TGG TGG TGC ACC          388                                                                       Val Phe Ala Trp Lys Gly Glu Thr Leu Gln Gl - #u Tyr Trp Trp Cys Thr                           110  - #               115  - #               120              - - GAG CGT GCC CTC GAC TGG GGC CCC GGC GGT GG - #C CCT GAC CTC ATC GTC          436                                                                       Glu Arg Ala Leu Asp Trp Gly Pro Gly Gly Gl - #y Pro Asp Leu Ile Val                       125      - #           130      - #           135                  - - GAT GAC GGC GGC GAC ACC ACT CTC TTG ATC CA - #T GAG GGG GTG AAG GCC          484                                                                       Asp Asp Gly Gly Asp Thr Thr Leu Leu Ile Hi - #s Glu Gly Val Lys Ala                   140          - #       145          - #       150                      - - GAG GAA GAG TAC GAG AAG ACG GGG AAG ATG CC - #C GAT CCG GCG TCT ACC          532                                                                       Glu Glu Glu Tyr Glu Lys Thr Gly Lys Met Pr - #o Asp Pro Ala Ser Thr               155              - #   160              - #   165                          - - GAC AAT GCT GAG TTC CAG ATC GTG CTC ACA AT - #C ATC AGG GAT GGG CTC          580                                                                       Asp Asn Ala Glu Phe Gln Ile Val Leu Thr Il - #e Ile Arg Asp Gly Leu           170                 1 - #75                 1 - #80                 1 -      #85                                                                              - - AAG GTG GAC CCC ACC AAG TAC AGG AAG ATG AA - #G GAT AGG ATT GTC        GGT      628                                                                    Lys Val Asp Pro Thr Lys Tyr Arg Lys Met Ly - #s Asp Arg Ile Val Gly                          190  - #               195  - #               200              - - GTG TCG GAG GAG ACC ACC ACC GGG GTC AAG AG - #G CTT TAC CAG ATG CAG          676                                                                       Val Ser Glu Glu Thr Thr Thr Gly Val Lys Ar - #g Leu Tyr Gln Met Gln                       205      - #           210      - #           215                  - - GCT AAC AAT TCC CTT CTT TTC CCT GCG ATC AA - #T GTC AAT GAC TCC GTC          724                                                                       Ala Asn Asn Ser Leu Leu Phe Pro Ala Ile As - #n Val Asn Asp Ser Val                   220          - #       225          - #       230                      - - ACC AAG AGC AAG TTT GAC AAT CTG TAT GGA TG - #C CGG CAC TCT CTT CCC          772                                                                       Thr Lys Ser Lys Phe Asp Asn Leu Tyr Gly Cy - #s Arg His Ser Leu Pro               235              - #   240              - #   245                          - - GAT GGT CTG ATG AGG GCC ACT GAT GTT ATG AT - #T GCT GGC AAG GTT GCA          820                                                                       Asp Gly Leu Met Arg Ala Thr Asp Val Met Il - #e Ala Gly Lys Val Ala           250                 2 - #55                 2 - #60                 2 -      #65                                                                              - - GTT GTC TGC GGT TAT GGT GAT GTC GGA GAG GG - #C TGT GCT GCT GCA        CTC      868                                                                    Val Val Cys Gly Tyr Gly Asp Val Gly Glu Gl - #y Cys Ala Ala Ala Leu                          270  - #               275  - #               280              - - AAG CAG GCT GGT GCC CGT GTT ATT GTG ACG GA - #G ATC GAC CCC ATC TGT          916                                                                       Lys Gln Ala Gly Ala Arg Val Ile Val Thr Gl - #u Ile Asp Pro Ile Cys                       285      - #           290      - #           295                  - - GCT CTT CAA GCC CTA ATG GAG GGT CTT CAG GT - #C CTC ACC CTC GAG GAT          964                                                                       Ala Leu Gln Ala Leu Met Glu Gly Leu Gln Va - #l Leu Thr Leu Glu Asp                   300          - #       305          - #       310                      - - GTT GTC TCA GAG GCG GAT ATC TTT GTT ACC AC - #C ACC GGT AAC AAG GAC         1012                                                                       Val Val Ser Glu Ala Asp Ile Phe Val Thr Th - #r Thr Gly Asn Lys Asp               315              - #   320              - #   325                          - - ATC ATC ATG CTG GAC CAC ATG AGG AAG ATG AA - #G AAC AAT GCC ATT GTC         1060                                                                       Ile Ile Met Leu Asp His Met Arg Lys Met Ly - #s Asn Asn Ala Ile Val           330                 3 - #35                 3 - #40                 3 -      #45                                                                              - - TGC AAC ATT GGT CAC TTT GAC AAC GAG ATT GA - #C ATG CTA GGT TTG        GAG     1108                                                                    Cys Asn Ile Gly His Phe Asp Asn Glu Ile As - #p Met Leu Gly Leu Glu                          350  - #               355  - #               360              - - ACA TAC CCT GGC ATC AAG AGA ATC ACC ATC AA - #G CCC CAG ACT GAC CGG         1156                                                                       Thr Tyr Pro Gly Ile Lys Arg Ile Thr Ile Ly - #s Pro Gln Thr Asp Arg                       365      - #           370      - #           375                  - - TGG GTC TTC CCT GAA ACC AAC ACT GGT ATA AT - #T GTT CTT GCT GAG GGC         1204                                                                       Trp Val Phe Pro Glu Thr Asn Thr Gly Ile Il - #e Val Leu Ala Glu Gly                   380          - #       385          - #       390                      - - CGA CTC ATG AAC CTT GGG TGT GCC ACT GGT CA - #C CCC AGC TTT GTC ATG         1252                                                                       Arg Leu Met Asn Leu Gly Cys Ala Thr Gly Hi - #s Pro Ser Phe Val Met               395              - #   400              - #   405                          - - TCC TGC TCC TTC ACC AAC CAG GTG ATT GCT CA - #G CTA GAG TTG TGG AAT         1300                                                                       Ser Cys Ser Phe Thr Asn Gln Val Ile Ala Gl - #n Leu Glu Leu Trp Asn           410                 4 - #15                 4 - #20                 4 -      #25                                                                              - - GAG AAG GCA AGC GGC AAG TAT GAG AAG AAG GT - #T TAC GTG CTC CCC        AAG     1348                                                                    Glu Lys Ala Ser Gly Lys Tyr Glu Lys Lys Va - #l Tyr Val Leu Pro Lys                          430  - #               435  - #               440              - - CAT CTT GAT GAG AAA GTA GCA GCG CTT CAC TT - #G GGC AAG CTC GGA GCC         1396                                                                       His Leu Asp Glu Lys Val Ala Ala Leu His Le - #u Gly Lys Leu Gly Ala                       445      - #           450      - #           455                  - - AAG CTT ACA AAG CTC AGC CCT TCA CAG GCG GA - #C TAC ATC AGC GTC CCC         1444                                                                       Lys Leu Thr Lys Leu Ser Pro Ser Gln Ala As - #p Tyr Ile Ser Val Pro                   460          - #       465          - #       470                      - - ATC GAG GGT CCC TAC AAG CCA CCT CAC TAC AG - #G TAC TAG ACGCTGTTGT          1493                                                                       Ile Glu Gly Pro Tyr Lys Pro Pro His Tyr Ar - #g Tyr  *                            475              - #   480              - #   485                          - - GCCGGGGAGA GATCATCGCA GCAAGAAAGT ATTAAGATTG AAGAAGAGAG TT -             #GTTATGGA   1553                                                                 - - GGACATGGCT ATATTTACTT TATTTCCTAC CTATTTCTTG CTGTTTCTCT TT -            #CCGAACTT   1613                                                                 - - TTAGACTGAT CCTCTTCTTC TCTTTGATTT ATTACGATAT GAATTCTGTT TA -            #AATTTTGC   1673                                                                 - - TTATTCTCTA ATGATGAGCT AGCAGACATA TGTTCTGTGG TAGAATAACG AG -            #GTTTTGAA   1733                                                                 - - CTTTGTGCAA AAAAAAAAAA AAAAAAAAAA AAAA       - #                  -     #      1767                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  485 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ala Leu Leu Val Glu Lys Thr Thr Ser Gl - #y Arg Glu Tyr Lys Val        1               5 - #                 10 - #                 15              - - Lys Asp Met Ser Gln Ala Asp Phe Gly Arg Le - #u Glu Ile Glu Leu Ala                   20     - #             25     - #             30                  - - Glu Val Glu Met Pro Gly Leu Met Ala Cys Ar - #g Ala Glu Phe Gly Pro               35         - #         40         - #         45                      - - Ala Gln Pro Phe Lys Gly Ala Lys Ile Thr Gl - #y Ser Leu His Met Thr           50             - #     55             - #     60                          - - Ile Gln Thr Ala Val Leu Ile Glu Thr Leu Th - #r Ala Leu Gly Pro Glu       65                 - # 70                 - # 75                 - # 80       - - Val Arg Trp Cys Ser Cys Asn Ile Phe Ser Th - #r Gln Asp His Ala Ala                       85 - #                 90 - #                 95              - - Ala Ala Ile Ala Arg Asp Ser Ala Ser Val Ph - #e Ala Trp Lys Gly Glu                  100      - #           105      - #           110                  - - Thr Leu Gln Glu Tyr Trp Trp Cys Thr Glu Ar - #g Ala Leu Asp Trp Gly              115          - #       120          - #       125                      - - Pro Gly Gly Gly Pro Asp Leu Ile Val Asp As - #p Gly Gly Asp Thr Thr          130              - #   135              - #   140                          - - Leu Leu Ile His Glu Gly Val Lys Ala Glu Gl - #u Glu Tyr Glu Lys Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Lys Met Pro Asp Pro Ala Ser Thr Asp As - #n Ala Glu Phe Gln        Ile                                                                                             165  - #               170  - #               175             - - Val Leu Thr Ile Ile Arg Asp Gly Leu Lys Va - #l Asp Pro Thr Lys Tyr                  180      - #           185      - #           190                  - - Arg Lys Met Lys Asp Arg Ile Val Gly Val Se - #r Glu Glu Thr Thr Thr              195          - #       200          - #       205                      - - Gly Val Lys Arg Leu Tyr Gln Met Gln Ala As - #n Asn Ser Leu Leu Phe          210              - #   215              - #   220                          - - Pro Ala Ile Asn Val Asn Asp Ser Val Thr Ly - #s Ser Lys Phe Asp Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Tyr Gly Cys Arg His Ser Leu Pro Asp Gl - #y Leu Met Arg Ala        Thr                                                                                             245  - #               250  - #               255             - - Asp Val Met Ile Ala Gly Lys Val Ala Val Va - #l Cys Gly Tyr Gly Asp                  260      - #           265      - #           270                  - - Val Gly Glu Gly Cys Ala Ala Ala Leu Lys Gl - #n Ala Gly Ala Arg Val              275          - #       280          - #       285                      - - Ile Val Thr Glu Ile Asp Pro Ile Cys Ala Le - #u Gln Ala Leu Met Glu          290              - #   295              - #   300                          - - Gly Leu Gln Val Leu Thr Leu Glu Asp Val Va - #l Ser Glu Ala Asp Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Phe Val Thr Thr Thr Gly Asn Lys Asp Ile Il - #e Met Leu Asp His        Met                                                                                             325  - #               330  - #               335             - - Arg Lys Met Lys Asn Asn Ala Ile Val Cys As - #n Ile Gly His Phe Asp                  340      - #           345      - #           350                  - - Asn Glu Ile Asp Met Leu Gly Leu Glu Thr Ty - #r Pro Gly Ile Lys Arg              355          - #       360          - #       365                      - - Ile Thr Ile Lys Pro Gln Thr Asp Arg Trp Va - #l Phe Pro Glu Thr Asn          370              - #   375              - #   380                          - - Thr Gly Ile Ile Val Leu Ala Glu Gly Arg Le - #u Met Asn Leu Gly Cys      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ala Thr Gly His Pro Ser Phe Val Met Ser Cy - #s Ser Phe Thr Asn        Gln                                                                                             405  - #               410  - #               415             - - Val Ile Ala Gln Leu Glu Leu Trp Asn Glu Ly - #s Ala Ser Gly Lys Tyr                  420      - #           425      - #           430                  - - Glu Lys Lys Val Tyr Val Leu Pro Lys His Le - #u Asp Glu Lys Val Ala              435          - #       440          - #       445                      - - Ala Leu His Leu Gly Lys Leu Gly Ala Lys Le - #u Thr Lys Leu Ser Pro          450              - #   455              - #   460                          - - Ser Gln Ala Asp Tyr Ile Ser Val Pro Ile Gl - #u Gly Pro Tyr Lys Pro      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Pro His Tyr Arg Tyr                                                                      485                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1847 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CTCGAGTGTT GACCTTTTCT GGTCGATTGA ATAGAATCGA ATGTCTTAAT CC -            #AGTACCCT     60                                                                 - - CCAGCTTTTA TTTCGTGTAA TTTATTTTCC AAACCTACCA CTACCAGTTT CA -            #TAACTCTC    120                                                                 - - GAATAAATTT ATCAAATAGT CTTTTGAGTG CTCAAAGTCT TGGGATAATA AA -            #TGGTCAGT    180                                                                 - - GCTATGTATC ACCCGGATGT GAAACATTAT GGGTGGAGAT AGACTATTAT AA -            #ATTTATTG    240                                                                 - - AAATATACGA TTGTTACTCG TTTAATAGCA AAAGTAGTAC AATGTATATA GT -            #TTCTATCG    300                                                                 - - AGAACAAGAT CTATTTAAAA TTCGAAAAGT ACATTTAAAA TTCATAAACA TA -            #TAAAGATA    360                                                                 - - GTAACATGTT AGATCTGCAT AGTACCACCA AAACAAGAAA AAAGAAACGC AC -            #ATCGCCAC    420                                                                 - - ATAATTGCTA TGATTCTCAC TGTCGGCTGC TTTGAAATAT TCGATTCTTT TG -            #GTAAATCA    480                                                                 - - CACAACATAA TATAATTACA ATAAATATAT ATATACTAAA GTATAATTAA TA -            #TAATTAAT    540                                                                 - - ACCACATTGT TTAATTCTGT TTTGATCTTT TAAGATCAGT CAGATCCACC GA -            #CGTTCCTA    600                                                                 - - CACGCGCAGG TCCAGATCCA AACAGCACAC ACACACACAC AATGCCACTA GT -            #GTAAATGC    660                                                                 - - TTGGTGGCTA TTGCATTTGC ACCTATTGAT ACTCTTTCTT CAAAAACAAG TT -            #ATTGTTTT    720                                                                 - - TATTTTCAAC CCAACTTTAA TACGGATTCA TACTGGGATT TAGGTGTTAA AT -            #CTGATAAT    780                                                                 - - TTAGGTTTGA ATAAGTTGTA TATTTGTTTC TTTGATTAAA AAAAGAACCT AT -            #ATATATAC    840                                                                 - - AAAAATAAAT AAAAAGTTCT AGATTTCAAT TTTCCGTATA TAGCGGGTTG AA -            #TTGTCTAT    900                                                                 - - TTTAATATGA AAATTGCGGA TCTTATAAAC AAAATGTTCT GAAATATGTA AA -            #AGGATTTA    960                                                                 - - GCCAAAGTTA ACCAAAAAAA AAAAAACAAA CAGAAAAGTC ACATTCACAT GT -            #CGTGGTAG   1020                                                                 - - ATCTAAGGCA TTAATTTAGA AATATGTCGT TACAATAAGC GGAGAACATG GG -            #ACGTTTCT   1080                                                                 - - CGTGGTCCAA TCAGACGAAC GAGATCTCAT AAATTAAATG ACTTCAGCGA GG -            #GAATTCAT   1140                                                                 - - GGCAGAATGA TAATGCAACT TAAGTGACTT TAGAGTGAAA ATGATACGAG AA -            #CAATGCAT   1200                                                                 - - AATCCATATG ACCGTTGAGT GAGTGATACC ATTAGCGCGA TACAAGCGGG AC -            #TATAAACT   1260                                                                 - - GATCTAGATT GTTTTTCTTG GGAAAAAATG TTACAAATTT TAAATATGTA GT -            #TTGAATTG   1320                                                                 - - TTAAACCAAG ATTCAACAGA AATATACCGT AAATAAACAA CAGTTGATAA TA -            #GTCATCGA   1380                                                                 - - AAAGATATCA ACTGATTCTT CACTTGGGCT ACTGTGACGG CCCGTTAGGT TC -            #TCAATATA   1440                                                                 - - AGTCAATAAC TACGATCTAC GATTCACTGA AACAAATAAA ACACAGCCAC GT -            #GTCCACCC   1500                                                                 - - TCCCACATCA CCGTCCGATC TAACCCACGA CAAGCTTACA ACACGGGTCA TA -            #CCGGCTCG   1560                                                                 - - TGCAGCGTGT TCCGTCATCC ACGGGATTAC AACTTCTACC AGATCCACCA AA -            #CCCTCAAA   1620                                                                 - - ACAATCTGAA CCGTTCATTT CATTTTGACC TCATCTATAT ATTCTCTGTC AC -            #TCCCCTTT   1680                                                                 - - CTCTTCTCCT CGCACACACT TCTCTCTCTC TCTCTCTCTG CCTCCTTTCG GA -            #TTCAAATC   1740                                                                 - - TCAGATCTAG CTCAACCATG GCGTTGCTCG TCGAGAAGAC CTCAAGTGGC CG -            #TGAATACA   1800                                                                 - - AGGTCAAAGA CATGTCTCAA GCCGATTTCG GTCGTCTCGA ACTCGAG   - #                  1847                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: ARABIDOPSIS - #SHH PROTEIN                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - His Ala Ala Ala Ala Ile Ala Arg Asp Ser Al - #a Ala Val Phe Ala Trp      1               5   - #                10  - #                15               - - Lys Gly Glu Thr Leu Gln Glu Tyr Trp Trp Cy - #s Thr Glu Arg Ala Leu                  20      - #            25      - #            30                   - - Asp Trp Gly Pro Gly Gly Gly Pro Asp Leu Il - #e Val Asp Asp Gly Gly              35          - #        40          - #        45                       - - Asp Ala Thr Leu Phe Arg Ile His Glu Gly Va - #l Lys Ala Glu Glu Ile          50              - #    55              - #    60                           - - Phe Glu Lys Thr Gly Gln Val Pro Asp Pro Th - #r Ser Thr Asp Asn Pro      65                  - #70                  - #75                  - #80        - - Glu Phe Gln Ile Val Leu Ser Ile Ile Lys Gl - #u Gly Leu Gln Val Asp                      85  - #                90  - #                95               - - Pro Arg Lys Tyr His Lys Met Lys Glu Arg Le - #u Val Gly Val Ser Glu                  100      - #           105      - #           110                  - - Glu Thr Thr Thr Gly Val Lys Arg Leu Tyr Gl - #n Met Gln Glu Asn Gly              115          - #       120          - #       125                      - - Thr Leu Leu Phe Pro Ala Ile Asn Val Asn As - #p Ser                          130              - #   135              - #   140                          - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: TOBACCO S - #HH PROTEIN                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - His Ala Ala Ala Ala Ile Ala Arg Asp Ser Ar - #g Ala Val Phe Ala Trp      1               5   - #                10  - #                15               - - Lys Gly Glu Thr Leu Gln Glu Tyr Trp Trp Cy - #s Thr Glu Arg Ala Leu                  20      - #            25      - #            30                   - - Asp Trp Gly Pro Gly Gly Gly Pro Asp Leu Il - #e Val Asp Asp Gly Gly              35          - #        40          - #        45                       - - Asp Ala Thr Leu Leu Ile His Glu Gly Val Ly - #s Ala Glu Glu Glu Tyr          50              - #    55              - #    60                           - - Ala Lys Ser Gly Lys Leu Pro Asp Pro Ser Se - #r Thr Asp Asn Val Glu      65                  - #70                  - #75                  - #80        - - Phe Gln Leu Val Thr Ile Ile Arg Asp Gly Le - #u Lys Thr Asp Pro Leu                      85  - #                90  - #                95               - - Lys Tyr Thr Glu Met Lys Glu Arg Leu Val Gl - #y Val Ser Glu Glu Thr                  100      - #           105      - #           110                  - - Thr Thr Gly Val Lys Arg Leu Tyr Gln Met Gl - #n Ala Asn Gly Thr Leu              115          - #       120          - #       125                      - - Leu Phe Pro Ala Ile Asn Val Asn Asp Ser                                      130              - #   135                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: BRACHYPODIUM - # SHH PROTEIN                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - His Ala Ala Ala Ala Ile Ala Arg Asp Ser Al - #a Ala Val Phe Ala Trp      1               5   - #                10  - #                15               - - Lys Gly Glu Thr Leu Glu Glu Tyr Trp Trp Cy - #s Thr Glu Arg Cys Leu                  20      - #            25      - #            30                   - - Asp Trp Gly Val Gly Gly Gly Pro Asp Leu Il - #e Val Asp Asp Gly Gly              35          - #        40          - #        45                       - - Asp Pro Thr Leu Leu Ile His Glu Gly Val Ly - #s Ala Glu Glu Glu Phe          50              - #    55              - #    60                           - - Glu Lys Ser Gly Lys Ile Pro Asp Pro Glu Se - #r Ala Asp Asn Pro Glu      65                  - #70                  - #75                  - #80        - - Phe Lys Ile Val Leu Thr Ile Ile Arg Asp Gl - #y Leu Lys Thr Asp Ala                      85  - #                90  - #                95               - - Arg Lys Tyr Arg Lys Met Lys Glu Arg Leu Va - #l Gly Val Ser Glu Glu                  100      - #           105      - #           110                  - - Thr Thr Thr Gly Ala Lys Arg Leu Tyr Gln Th - #r Gln Asn Pro Gly Thr              115          - #       120          - #       125                      - - Leu Leu Phe Pro Ala Ile Asn Val Asn Asp Se - #r                              130              - #   135                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: WHEAT SHH - # PROTEIN (1)                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - Arg Ala Ala Ala Ala Ile Ala Arg Asp Ser Al - #a Ser Val Phe Ala Trp      1               5   - #                10  - #                15               - - Lys Gly Glu Thr Leu Gln Gly Tyr Trp Trp Cy - #s Thr Glu Arg Ala Leu                  20      - #            25      - #            30                   - - Asp Trp Gly Pro Gly Gly Gly Leu Asp Leu Il - #e Val Asp Asp Gly Gly              35          - #        40          - #        45                       - - Asp Thr Thr Leu Leu Ile His Glu Gly Val Ly - #s Ala Glu Glu Glu Tyr          50              - #    55              - #    60                           - - Glu Lys Thr Gly Lys Met Pro Asp Pro Thr Se - #r Thr Asp Asn Ala Glu      65                  - #70                  - #75                  - #80        - - Phe Gln Ile Val Leu Thr Ile Ile Arg Asp Gl - #y Leu Lys Val Asp Pro                      85  - #                90  - #                95               - - Thr Lys Tyr Arg Lys Met Lys Asp Arg Ile Va - #l Gly Val Ser Glu Glu                  100      - #           105      - #           110                  - - Thr Thr Thr Gly Val Lys Arg Leu Tyr Gln Me - #t Gln Ala Asn Asn Ser              115          - #       120          - #       125                      - - Leu Leu Phe Leu Thr Ile Asn Val Asn Asp Se - #r                              130              - #   135                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: WHEAT SHH - # PROTEIN (2)                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - Gln Ala Ala Ala Ala Ile Ala Ala Ala Gly Il - #e Pro Val Phe Ala Trp      1               5   - #                10  - #                15               - - Lys Gly Glu Thr Glu Glu Glu Tyr Glu Trp Cy - #s Ile Glu Gln Thr Ile                  20      - #            25      - #            30                   - - Leu Lys Asp Gly Lys Pro Trp Asp Ala Asn Me - #t Val Leu Asp Asp Gly              35          - #        40          - #        45                       - - Gly Asp Leu Thr Glu Ile Leu His Lys Lys Ty - #r Pro Gln Met Leu Glu          50              - #    55              - #    60                           - - Arg Ile His Gly Ile Thr Glu Glu Thr Thr Th - #r Gly Val His Arg Leu      65                  - #70                  - #75                  - #80        - - Leu Asp Met Leu Lys Ala Gly Thr Leu Lys Va - #l Pro Ala Ile Asn Val                      85  - #                90  - #                95               - - Asn Asn Ala                                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - GCGTCTAGAT GCAACATMTT CTCMACYCAG GA       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - GCGTCTAGAT TRTCAAACTT GCTCTTGGTR AC       - #                  - #              32                                                                    __________________________________________________________________________

We claim:
 1. An isolated promoter derived from theS-adenosyl-L-homocysteine hydrolase gene of A. thaliana.
 2. An isolatedpromoter having the nucleotide sequence of that part of SEQ ID NO 3which is upstream of the methionine codon as shown in FIGS. 5A-5C.
 3. ADNA construct comprising a promoter as claimed in claim 1 or claim 2operably linked to a gene.
 4. A DNA construct as claimed in claim 3wherein said gene is selected from the group consisting of a geneencoding S-adenosyl-L-homocysteine hydrolase, an antifungal proteingene, a selectable marker gene, NptII, a kanamycin resistance gene, aphosphinothricin resistance gene, the phosphinothricin acetyltransferase (PAT) gene, the glucuronidase (GUS) reporter gene, and theluciferase (LUC) reporter gene.
 5. A vector comprising a DNA constructas claimed in claim 3 or claim
 4. 6. A vector as claimed in claim 5which is a binary agrobacterium vector or a direct DNA delivery vector.7. A plant cell transformed with a vector as claimed in claim
 5. 8. Agenetically transformed plant or part thereof having stably incorporatedinto the genome the DNA construct as claimed in claim 3 or claim
 4. 9. Aplant cell or genetically transformed plant according to claim 7 orclaim 8, wherein the plant is wheat, maize, oil seed rape, potato,tomato, banana or tobacco.
 10. A method of increasing the resistance ofa plant to infection by a pathogenic organism, the method comprisingtransforming the plant with a vector comprising a promoter according toclaim 1 or claim 2 operably linked to a gene conferring resistance tothe pathogenic organism.
 11. A method as claimed in claim 10 wherein thepathogenic organism is a fungus and the gene encodes an antifungalprotein.
 12. A genetically transformed plant or part thereof accordingto claim 8 which is a cell, protoplast or seed.